Mycobacterium tuberculosis survives, multiplies, and, persists in infected macrophages. In this proposal, we plan to study oxidative stress response defenses in M. tuberculosis, and determine how the tubercle bacillus withstands, eludes, and interferes with systems generating reactive oxygen and nitrogen intermediates (ROI and RNI) in the host phagocytic cells. Our studies have originated from early observations that the M. tuberculosis genome has a defective gene (pseudogene) encoding a regulator of oxidative stress response, and that the expression of anti-oxidant systems is anomalous in the virulent M. tuberculosis strains. These studies have now transformed into a two-prong study of (a) systems in M. tuberculosis that detoxify ROI and RNI (with added implications for isoniazid action and the exquisite sensitivity of the tubercle bacillus to INH); and (b) the previously unappreciated mechanisms of interference with the assembly, localization, or activation of enzymatic systems responsible for the delivery of bactericidal oxygen and nitrogen intermediates into the mycobacterial phagosome. Several new observations provide the foundation for this application: (a) New evidence shows that INH activation with KatG results in the generation of NO*, known to show strong anti-M, tuberculosis action. (b) The observed alterations of the mycobacterial phagosome suggest that M. tuberculosis interferes with the assembly or activation of phagocytic NADPH oxidase via interference with phosphatidylinositol phosphate interconversions on mycobacterial phagosomes. (c) The preliminary data also show that inducible nitric oxide synthase (iNOS) is excluded from the vicinity of mycobacterial phagosome. We propose the following 3 specific aims to demonstrate the relationships listed above: 1) Investigate the unusual adaptations of M. tuberculosis oxidative stress response systems. 2) Investigate the mechanism of NO* generation during INH activation by KatG and its role in antimycobacterial action of isoniazid. 3) Investigate molecular mechanisms of M. tuberculosis interference with iNOS recruitment to mycobacterial phagosomes and with oxidative bactericidal mechanisms in macrophages. We anticipate that these findings will explain important aspects of M. tuberculosis pathogenesis and its ability to parasitize macrophages. The phenomena addressed in this proposal will provide a foundation for development of new therapies that will improve present treatments of tuberculosis.